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INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1995, p. 565-571 0020-7713/95/$O4.00+0 Copyright 0 1995, International Union of Microbiological Societies Vol. 45, No. 3 Lactosphaera gen. ~ o v . a, New Genus of Lactic Acid Bacteria, and Transfer of Ruminococcus pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov. PETER H. JANSSEN,’r2* STEFAN EVERS,3 FREDERICK A. RAINEY,2p4NORBERT WEISS,4 WOLFGANG LUDWIG,3 CHRIS G. HARFOOT,2 AND BERNHARD SCHINK’ Fakultat f i r Biologie, Universitat Konstanz, D- 78434 Konstanz, Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-80333 Munich, and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 0-38124 Braunschweig, Germany; and Department of Biological Sciences, University of Waikato, Hamilton, New Zealand2 The phylogenetic position and physiology of strain KoTa2* (T = type strain), which was previously classified as a Ruminococcuspasteurii strain, were studied. A determination of the 16s ribosomal DNA sequence of this taxon revealed its position within the radiation of the gram-positive lactic acid bacteria having low DNA G+C contents and that it is closely related to the genus Curnobucterium.L-Lactic acid was produced from glucose by a fructose-1,6-bisphosphate-activated lactate dehydrogenase, and oxygen tolerance was observed, characteristics which are consistent with assignment to this group. On the basis of its phenotypic characteristics and unique signature nucleotides, we propose that strain KoTa2 (= DSM 2381 = ATCC 35945) should be transferred to a new genus, Lactosphueru gen. nov., as the type strain of the speciesLactosphaera pmteurii comb. nov. The species Ruminococcus pasteurii was originally described to encompass L-tartrate-fermenting anaerobic cocci (27). This bacterium is able to ferment L-tartrate, citrate, oxaloacetate, pyruvate, and a variety of sugars, and the fermentation products have been reported to be acetate, formate, ethanol, and carbon dioxide (27). On the basis of the following characteristics of strain KoTa2= (T = type strain) (27), the original assignment of this organism to the genus Ruminococcus seemed to be justified: morphology, the fermentation end products, and the G + C content of the DNA. However, our investigations showed that the type strain of R pasteurii, strain KoTa2, is not physiologically or genetically related to other members of the genus Ruminococcus. The production of significant amounts of lactic acid, aerotolerance, and the results of a 16s ribosomal DNA (rDNA) sequence analysis indicated that this strain, previously classified as a R. pasteurii strain, belongs to a new taxon of lactic acid bacteria. Therefore, we describe a new genus, Lactosphaera, and designate the type strain of R. pasteurii, strain KoTa2 (= DSM 2381), the type strain of a new species, Lactosphaera pasteurii comb. nov. was adjusted to the appropriate value with NaOH. Acetate agar (which contained 15 g of agar per liter) was prepared as described by Atlas and Parks (1). Lactic acid production and metabolic studies. High-performance liquid chromatography and gas chromatography with a thermal conductivity detector were performed as described previously (17). The isomeric form of lactic acid was determined enzymatically (2) by using D- and L-lactic acid dehydrogenases (LDHs) (Boehringer, Mannheim, Germany). Growth yields were determined as described previously (16). Fructose-l,6-bisphosphate(Fru-1,6-P,)-activated LDH activity was measured at 34°C as described elsewhere (21). Fru-l,6-P2-independent LDH activity was measured by the same method except that Fru-1,6-P2 was omitted. Further characterization of strain K o T was ~ ~ carried ~ out by using previously described methods (15-17,25). Peptidoglycan analysis. Cell walls were prepared and the peptidoglycan structure was determined by the methods of Schleifer and Kandler (28), modified by using thin-layer chromatography on cellulose sheets instead of paper chromatography. Briefly, 1 mg of freeze-dried cell wall material was hydrolyzed in 0.2 ml of 4 M HCl at 100°C for 16 h (total hydrolysate) or 45 min (partial hydrolysate). The diamino acids in the total hydrolysate were identified by one-dimensional chromatography by using methanol-pyridine-water-10 M HCI (32:4:7:1, vol/vol/ volhrol). The amino acids and peptides in the partial and total hydrolysates were identified by their mobilities and staining characteristics with ninhydrin spray after two-dimensional chromatography (28). The resulting “fingerprints” were compared with those of known peptidoglycan structures. Oxygen tolerance. Oxygen was added to the desired concentration with a syringe to sealed 120-ml serum vials containing (under an N,-CO, [80:20] atmosphere) 50 ml of anoxic medium supplemented with 10 mM sodium L-tartrate and 2 mM Na2S20, but no sulfide reductant. Hemin and hematin were added from filter-sterilized (pore size, 0.2 p,m) stock solutions in 2% methylamine, so that the final methylamine concentration was 0.9 mM. Catalase (29) and superoxide dismutase (33) activities were assayed by using cell extracts that were prepared by French press treatment under anoxic conditions (18). 16s rDNA analysis. Purification of genomic DNA, in vitro amplification of 16s rRNA genes, and a direct sequence analysis of amplified DNA fragments were performed with strain KoTa2= and R. JIavefaciens as described elsewhere (26, 30). The derived 16s rRNA primary structure of strain KoTa2= was added to an alignment of about 1,800 homologous sequences from bacteria. The phylogenetic affiliation of strain K o T was ~ ~determined ~ by using distance matrix and maximum-parsimony methods and a data set containing all of the available 16s rRNA sequences from gram-positive bacteria having low genomic DNA G+C contents. Subsets of the data were analyzed by using a maximum-likelihood-based treeing procedure. The reference sequences used were obtained from public databases (22, 24). The complete sequences were used to analyze close relationships, whereas the more variable sequence positions (positions at which the sequences were invariant in less than 50% of the sequences in the entire data set) were deleted to investigate remote relationships. The data analyses were performed by MATERIALS AND METHODS Strain and media. Strain K o T ~ which ~ ~ , was originally described as a R. pasteurii strain (27), was obtained from our collection. RurninococcusJlavefaciens C94 (= ATCC 19208) was purchased from the American Type Culture Collection, Rockville, Md. A freshwater mineral medium supplemented with vitamins (27) was used to cultivate strain K o T ~ Yeast ~ ~ . extract was added at a concentration of 0.02% (wt/vol) unless noted otherwise. Substrate tests were performed by using sugars (D isomers) at an initial concentration of 2 mM and organic acids (L isomers) at a concentration of 10 mM. Polysaccharides were added at a concentration of 0.1% (wt/vol). All cultures were incubated at 34°C. The buffers 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-(2-hydroxyethyl)-piperazine-N’-(3-propanesulfonic acid) (EPPS) were purchased from Sigma Chemical Co.,St. Louis, Mo.,and the pH of each medium * Corresponding author. Present address: Max-Planck-Institut fiir Terrestrische Mikrobiologie, D-35043 Marburg, Germany. Fax: (49) 6421 161470. 565 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:51:33 566 INT.J. SYST.BACTERIOL. JANSSEN ET AL. TABLE 1. Overall levels of similarity (values at lower left) of the 16s rRNA primary structures of strain K o T ~Carnobacterium ~ ~ , spp., selected lactic acid bacteria, and other bacteria, including members of the genus Rurninococcus % Similarity" Organism Strain K o T ~ ~ ~ Camobacterium alterjknditum Camobactenum divergens Camobacterium funditum Camobacterium gallinarum Camobacterium mobile Carnobacteriumpiscicola Enterococcus faecalis Vagococcusfluvialis Lactococcus lactis Streptococcus thermophilus Bacillus subtilis Ruminococcus flavefaciens Ruminococcus gnavus Ruminococcus hansenii Ruminococcus productus Ruminococcus torques Escherichia coli Strain K0'l'a2~ 94.6 94.1 94.2 94.3 95.1 94.2 92.0 92.6 87.2 87.0 91.2 83.0 76.1 75.2 78.7 79.1 78.4 Camobacterium alterjknditum Camobacterium divergens Camobacterium funditurn Carnobacterium gallinamm Camobacterium mobile Carnobacterium piscicola 95.6 95.0 96.5 95.6 97.2 96.5 95.0 96.1 96.9 96.1 95.1 96.5 96.1 97.7 96.0 95.2 96.5 97.2 96.5 98.2 96.3 95.3 95.9 94.9 96.5 95.2 92.6 92.1 87.8 87.5 90.3 81.1 75.8 74.8 78.1 79.1 78.4 95.5 96.7 96.3 96.9 92.6 92.5 87.0 88.2 90.2 81.7 75.8 75.5 78.6 79.2 78.1 95.O 97.7 95.4 93.8 91.9 87.1 87.6 90.0 81.7 75.9 75.1 77.5 78.9 77.5 96.1 98.0 92.1 91.1 86.2 87.3 90.3 81.1 74.7 74.5 77.3 78.2 77.4 96.4 93.6 93.3 87.3 87.4 90.1 81.5 76.1 75.7 78.5 79.7 78.0 92.0 91.8 86.3 87.5 90.3 81.3 75.4 75.0 78.0 78.8 77.5 ~~ ~ 'The values on the upper right were calculated by using only data for positions that had been unambiguously determined for all members of the genus Camobacterium. Peptidoglycan structure. Purified cell walls of strain K o T ~ ~ ~ contained the amino acids lysine, glutamic acid, aspartic acid, and alanine at a molar ratio of 1:1:1:2, The fingerprints of the partial hydrolysate and the presence of the hydrolysis-stable compound E-( aminosuccinyl-)lysine are compatible only with peptidoglycan type A4a, L-LYs-D-As~. RESULTS Oxygen tolerance. Strain K o T was ~ ~able ~ to grow in mineral medium containing 10 mM L-tartrate but no sulfide re16s rDNA sequence analysis. Almost complete 16s rRNA ductant (with 2 mM Na,S,O, added as a sulfur source) in the genes of strain K o T and ~ ~R.~Jlavefuciens were amplified in absence of yeast extract. Addition of 2.5% (vol/vol) 0, to the vitro and sequenced directly. Different treeing methods and headspace of static cultures inhibited growth, but this inhibidifferent data sets were used to reconstruct the phylogenetic tion was overcome by the addition of yeast extract. Strain relationships of strain K o T ~Strain ~ ~ .K o T is~only ~ distantly ~ K o T grew ~ ~well ~ on unreduced medium supplemented with 2 related to members of the genus Ruminococcus (4,6,7), but is mM Na,S,O, and 0.02% (wt/vol) yeast extract in the presence closely related to members of the genus Curnobacterium(9,11, of 0, partial pressures up to 10% (volhol). When the 0, 34). The overall levels of sequence similarity for strain partial pressure was between 10 and 16% (vol/vol), growth was K o T ~members ~ ~ , of the genus Curnobucterium,selected lactic poor, while no growth occurred when the 0, partial pressure acid bacteria, and other bacteria are shown in Table 1. The close phylogenetic relationship between strain K o T and ~ ~ ~ was greater than 18% (vol/vol). In the presence of 5% (vol/vol) O,, the amount of acetate produced from L-tartrate was not members of the genus Curnobacterium is shown in Fig. 1. The significantly changed, but a decrease in formate formation was position of this group among the lactic acid bacteria is shown detected (Table 2). There was no significant increase in the in Fig. 2. True members of the genus Ruminococcus are inspecific growth yield in the presence of oxygen. cluded in Fig. 2 to show the great phylo enetic distance between these organisms and strain KoTa2$. Addition of 10 pM hemin or 10 pM hematin (dissolved in methylamine so the final methylamine concentration was 0.9 mM) or addition of 10 p,M MnCl, did not overcome the growth inhibition caused by the presence of 2.5% (vol/vol) 0, C.gallinarum in the headspace in the absence of yeast extract. Methylamine Cdivergens at a concentration of 0.9 mM did not inhibit growth in the presence of 2.5% (vol/vol) 0, when the medium contained 0.02% (wt/vol) yeast extract. No superoxide dismutase or catalase activity was observed in cell extracts prepared from cells grown in the presence of 2.5% (vol/vol) 0, with 10 mM Ltartrate and 0.02% (wt/vol) yeast extract. However, growth occurred in the presence of air on Trypticase agar plates (12). B. subtilis Carbon sources that support growth. Mannitol, sorbitol, and FIG. 1. Phylogenetic tree derived from 16s rDNA sequence analysis, reflectgalactose supported growth of strain K o T ~while ~ ~ ribose , and ing the relationships of strain KoTa2= and members of the genus Camobacteglycerol did not. Strain KoTa2= grew on starch and oat spelt rium. The tree was reconstructed by using a maximum-likelihood method. Bar = xylan and grew weakly on laminarin. The following compounds 10% estimated sequence divergence. Abbreviations: B., Bacillus; C., Camobacterium. did not support growth: chitin, gum karaya, carboxymethyl using the ARB program package (23) and the treeing programs NEIGHBOR (8) and fastDNAml (22). Nucleotide sequence accession numbers. The nucleotide sequences of strain K o T and ~ R. ~ flavefuciens ~ have been deposited in the EMBL data library under accession numbers X85097 and X87150, respectively. . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:51:33 VOL. 45, 1995 LACTOSPHAERA GEN. NOV. \ 567 Streptococcus Leuconostoc, Weissella nterococcus I Carnobacterium E. coli R . jlavefaciens FIG. 2. Consensus tree showing the phylogenetic positions of strain K o T and ~ ~the~genus Curnobacterium among the lactic acid bacteria and the distances between these taxa and the genus Ruminococcus. The tree was based on the results of maximum-likelihood analyses and was corrected by using the results of distance matrix and maximum-parsimony analyses. Only alignment positions which are occupied by identical nucleotides in at least 50% of all available 16s rRNA sequences from gram-positive bacteria with low DNA G+C contents were included. The triangles indicate phylogenetic groups. Bar = 10% estimated sequence divergence. Abbreviations: E., Escherichia; R., Ruminococcus. cellulose, amorphous cellulose, mannan, lichenan, carrageenduced (as a proportion of the total products) increased as the an, gum locust bean, pullulan, arabinogalactan, and glycogen. acidity of the growth medium increased (Fig. 3). Fermentation of glucose. Strain K o T produced ~ ~ ~ L-lactate The LDH activity was higher in cultures grown at more (in addition to formate, acetate, and ethanol) from a wide acidic pH values than in cultures grown at more basic pH range of sugars, including glucose, fructose, maltose, lactose, values (Table 4). The LDH activity was very much lower (0 to sucrose, cellobiose, and sorbitol. Lactate was not formed from 5%) if Fru-1,6-P2 was omitted from the enzyme assay mixture. Cultures grown on L-tartrate or pyruvate did not produce lacL- tartrate, pyruvate, or citra te . tate. Cells grown on pyruvate contained a Fru-1,6-P2-activated Strain K o T was ~ ~grown ~ on 15 mM glucose, and the end products of fermentation were determined (Table 3). SignifiLDH activity, and this activity was higher in cells grown under more acidic conditions than in cells grown under more basic cant quantities of lactate were produced (65% of the total conditions . product carbon), but very little hydrogen was produced. During growth the pH decreased from 7.2 to 5.5. The increase in Additional characteristics of strain K o T ~ Esculin ~ ~ . was lactate production was apparently not a consequence of the hydrolyzed. Hydrogen in the headspace (H,-CO,, 80:20) did not inhibit growth on L-tartrate. Fumarate was not reduced. No inclusion of yeast extract in the growth medium. The amount growth occurred in the presence of 3% (wthol) NaC1, but of lactate produced did not change when the level of yeast growth did occur in the presence of 2% (wthol) NaC1. No extract in the medium was varied from 0.01 to 0.1% (wthol) in the presence of 100 pg of biotin per liter. Strain K o T also ~ ~ ~ growth occurred on acetate agar (pH 5.4). produced the same amount of lactate in the absence of yeast extract. DISCUSSION Lactate production in the presence of a range of pH values. 16s rDNA sequence analysis. Our comparative 16s rDNA Strain K o T was ~ ~grown ~ on 4 mM glucose at different pH sequence analyses revealed that strain K o T is~most ~ ~closely values by using heavily buffered media. The pH did not change related to the species of the genus Carnobacterium and is not during fermentation of glucose. The amount of lactate pro- TABLE 2. Fermentation balances of strain KoTa2= grown on L-tartrate in the absence and presence of oxygen" O2 concn in gas phase (%I 0 5 Amt of L-tartrate degraded (Fmol) 500 500 Amt of products (Fmol) Formate Acetate Amt of cell matter (mgY 426 166 441 409 6.66 6.71 Amt of substrate assimilated (FmW 110 111 % Carbon recovery" Ratio of formate to acetate 106.9 88.3 0.97 0.41 The data are the means of the results of two independent experiments and were not corrected for the products and cell matter produced on yeast extract. Cell dry weights were calculated from culture densities (optical densities at 650 nm) by using conversion factors which were obtained by direct gravimetric determinations performed with 1-liter cultures. Calculated by using <C,H,O,> as the empirical formula for cell matter. Calculated by assuming that 1 mol of C 0 2 was produced per mol of L-tartrate degraded; the C 0 2 produced from HCOO- was not included in the calculation. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:51:33 568 INT.J. SYST.BACTERIOL. JANSSEN ET AL. TABLE 4. Fru-1,6-P2-activatedLDH activities in strain KoTa2' grown on glucose and pyruvate at various pH values TABLE 3. Fermentation balance of strain KoTa2 grown on 15 m M glucose" Carbon balance Substrate or product Glucose Lactate Formate Acetate Ethanol H2 cop Cells" Amt (IJJnol) Hydrogen balance Redox value hmol)c Fmol of carbonb Amt of available H (Pmol)b 4,500 2,601 343 422 646 0 191 472 18,000 10,404 686 1,688 3,876 0.2 0 2,006 750 867 343 211 323 0.1 191 118 0 0 +343 0 -646 -0.2 +382 -59 % of product Substrate Growth PH Buffer carbon as lactate Fru-1,6-P,-activated LDH activity (pmol. min-' mg of protein-')a Glucose 6.2 7.1 8.0 6.2 7.1 8.0 MES MOPS EPPS MES MOPS EPPS 74 37 26 0 0 0 0.419 0.459 0.113 0.127 0.129 0.011 Pyruvate - a The Fru-1,6-P2-independent LDH activity was less than 5% of the Fru-1,6P2-activatedLDH activity in all cases. "The data are the means of the results of two independent experiments performed with 50-ml cultures in the presence of 0.1% (wthol) yeast extract. The values were corrected for the products and cell matter (9 mg liter-') produced on 0.01% (wthol) yeast extract. The balance was 104%. The balance was 1.03 pmol. The amount of C 0 2 was estimated (because we used a bicarbonate-buffered medium) as follows: micromoles of C 0 2 = millimoles of acetate + micromoles of ethanol - micromoles of formate. Cell dry weights were calculated from culture densities (optical densities at 650 nm) by using conversion factors which were obtained by direct gravimetric determinations performed with 1-liter cultures. Values were calculated by using <C,H,O,> as the empirical formula for cell matter. which we obtained are shown in Table 1 and support the congeneric status of the Carnobacterium spp. (similarity values, 96.0 to 98.2%) and the exclusion of strain K o T (similarity ~ ~ ~ values, 95.0 to 95.6%). The separation of these taxa is also reflected by the primary structure signatures. There are a number of residues in the 16s rRNA primary structure of strain KoTa2= that differ from the residues in the Carnobacterium consensus sequence (Table 5), allowing strain K o T to~ be ~ ~ differentiated from members of the genus Camobacterium. The tree in Fig. 1 shows the phylogenetic unity of the genus Carnobacterium and the somewhat remote position of strain related to members of the genus Ruminococcus. The data K o T ~ This ~ ~ tree . was reconstructed by using a maximumindicated that the Carnobacterium species can be considered a likelihood method, and all alignment positions were included phylogenetic unit that does not include strain K o T ~ The ~ ~ . for the calculations. The 16s rRNA sequence of Bacillus sublevels of intrageneric similarity range from 94.9 to 98.0%, while tilis, a moderately closely related outgroup reference organism, the corresponding values for strain K o T and ~ ~Carnobacte~ was used to root the tree. The topology of the tree was evalrium species are 94.1 to 95.1%. There is a slight overlap of uated by using alternative treeing methods and various data values because of the high similarity value obtained for strain sets that differed with respect to alignment positions and refK o T and ~ ~Camobacterium ~ mobile. However, most of the erence organisms. The branching pattern was stable in most sequences of the Camobacterium species are incomplete at cases. The tree in Fig. 2 shows the position of the strain their 3' ends, and the sequence data for C. mobile do not K0Ta2~-Camobacteriurncluster among the major groups of include data for the 5'-terminal portion. Therefore, we also lactic acid bacteria. This tree is a consensus tree based on the calculated similarity values based on only the homologous poresults of a maximum-likelihood analysis that included 16s sitions determined for all Carnobacterium spp. The values rRNA sequences from all members of the cluster, selected TABLE 5. Signature nucleotides in the 16s rRNA primary structure of strain K o T which ~ ~ are ~ different from the nucleotides in the consensus sequence of the genus Carnobacterium 80 - Nucleotide(s) in: Position(s)" Strain KoTa2= 60 40 - 6 7 8 PH FIG. 3. Lactate production from glucose by strain KoTdT at ditferent pH MOPS buffer; A, EPPS buffer. All buffers values. Symbols: 0, MES buffer; 0, were used at a concentration of 100 mM and were adjusted to the required pH with NaOH. 69 156 166 210 316 * 337 443 * 491 456 457-458 * 474-475 592 * 647 614 * 626 846 1152 1256 1278 1482 a E. coli numbering system (3). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:51:33 G C G U U-A C-G U UG-CA C-G A*U A A C G G Camobacterium consensus sequence C U A c C-G U*A G AU AU U-A C-G G G U U A - VOL.45, 1995 LACTOSPHAERA GEN. NOV. 569 TABLE 6. Characteristics that distinguish strain KoTa2= from the genera Rurninococcus and Cumobucterium Taxon Typical morphology E?; Growth content (mo]%) on cellu'ose Lactic acid -h 39-46 + or -= - - 45 33-37 - Peptidoglycan type R U ~ ~ ~ ~ O C O CCocci C U Sor~ oval Aly, rn-Dpm-direct cells with pointed ends Cocci Strain KoTa2= Cumobucteriume Short to mediumlength rods A4a, L-LYS-D-ASP Aly, m-Dpm-direct + or -' Major products +d + Oxygen Minimum growth temp ("C) + - 20-30'" - + + 0 0 Succinic acid Hydrogen + o r -' - Data from references 4, 7, and 35. -, negative; +, positive. Strain or species dependent. Production of lactic acid depends on the pH of the culture. Data from references 5, 9, 11, and 14. representatives of the major lines of lactic acid bacteria, members of the genus Ruminococcus, and Escherichia coli as an outgroup reference organism. Given that lower levels of relationships had to be resolved, the data for variable alignment positions (positions at which the sequences were invariant in less than 50% of the sequences in the entire data set) were deleted from the data set. The tree was corrected on the basis of the results of distance matrix and maximum-parsimony analyses of about 550 16s rRNA sequences from gram-positive bacteria with low DNA G+C contents. The results of the majority of the analyses supported common roots for the genera Vagococcus and Enterococcus and the genera Lactococcus and Streptococcus. However, the branching order of these groups and the remaining major groups of lactic acid bacteria could not be determined unambiguously. This was indicated by multiple furcations in the tree. The deep branching of the genus Ruminococcus was supported by the results of all of the analyses which we performed. Lactic acid production. Strain K o T formed ~ ~ ~L-lactate, as well as forrnate, acetate, and ethanol, from glucose in a mixed acid type of fermentation. Up to 2.0 mol of lactate was produced per mol of glucose fermented, depending on the growth pH. A shift to lactate production from acetate and ethanol production with increasing acidity has been reported in a variety of microorganisms (31), and this shift is mediated by a Fru-1,6-P2-regulated LDH (10, 13). The Fru-1,6-P2 activation of LDH activity explains why lactate was not produced when the organism was grown on pyruvate, since Fru-1,6-P2, is not an intermediate of pyruvate catabolism. In pyruvate-grown cells the Fru-1,6-P2-dependent LDH activity increased as the acidity of the medium increased, although no lactate was produced. Thus, pH appears to control induction of this enzyme, while the enzyme activity is allosterically regulated by Fru-1,6p2* Oxygen tolerance. Strain K o T could ~ ~ grow ~ in the presence of yeast extract under microoxic to oxic conditions, but did not contain catalase or superoxide dismutase activities. Addition of hemin, hematin, or MnCl, (all at a concentration of 10 FM) did not allow the organism to grow microaerobically in the absence of yeast extract; such additions are known to be components of catalase and pseudocatalase (20, 36). The basis of the microaerotolerance of strain K o T remains ~ ~ ~ to be explained, and the role of yeast extract in conferring oxygen tolerance needs to be elucidated. Strain K o T apparently ~ ~ ~ gained no additional metabolic energy during growth in the presence of oxygen; there were no significant changes in acetate production or specific growth yield. The amount of formate produced during growth on L-tartrate in the presence of oxygen was greatly reduced. Transfer of electrons to oxygen by lactic acid bacteria has been described previously (11, 32), and such a transfer can produce superoxide or H,02. No superoxide dismutase activity has been found in the lactic acid bacteria, and although members of the genus Lactobacillus can have hematin-containing catalases (36, 37) or manganese-containing pseudocatalase activities (19), members of the genus Carnobacterium are catalase negative (1l).Lactic acid bacteria can be oxygen tolerant, and members of the genus Carnobacterium can even grow in the presence of air (11). In contrast, Rurninococcus spp. are strict anaerobes (4, 7). In this respect, strain K o T is~ physiologically ~ ~ similar to the lactic acid bacteria, particularly members of the genus Carnobacterium,and is not like members of the genus Ruminococcus. Distinguishing features of strain K o T ~ Strain ~ ~ . K o T ~ ~ ~ can be distinguished from members of the genus Ruminococcus by the fact that it produces significant amounts of lactate under acidic conditions, by the fact that it does not produce significant amounts of hydrogen or succinate from glucose, by its oxygen tolerance, and by other characteristics (Table 6). Our comparative sequence analysis of the 16s rRNA gene showed that strain K o T is~ not ~ related ~ to members of the genus Ruminococcus and is closely related to members of the genus Carnubactenurn. Strain K o T can ~ ~be~ distinguished from members of the genus Carnobacteriumby its coccal morphology and DNA base composition (45 mol% G+C) (Carnobacterium spp. have short to medium-length, straight, slender, rod-shaped cells and DNA base compositions ranging from 33 to 37 mol% G+C) (Table 6) and by its unique signature nucleotides (Table 5). The peptidoglycan type of all previously described members of the genus Carnobacterium is type Aly, m-Dpm-direct (5, 9, 14), while the peptidoglycan type of strain K o T is~ type ~ ~A4a, L-LYS-D-ASP. On the basis of the data described above, we propose that strain KoTa2, previously the type strain of R. pasteurii, should be reclassified in a new genus, Lactosphaera gen. nov., as the type strain of Lactosphaera pasteurii comb. nov. The description below is based on data from this study and a previous study (27). Description of Lactosphaera gen. nov. Lactosphaera (Lac.to. sphae'ra. L. gen. n. Zactis, milk [used because of its association with lactic acid fermentation]; Gr. fem. n. sphaira, a sphere; N.L. fem. n. Lactosphaera, a lactic acid-producing sphere). Lactosphaera cells are gram-positive, nonmotile, nonsporulating cocci. Aminopeptidase negative. The cell wall is a single thick layer that is about 25 nm thick. The peptidoglycan type is type A4a, L-L~s-D-As~. Fermentative chemoorganotrophic metabolism. Some organic acids (metabolized via pyruvate) and sugars are used as Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 16:51:33 570 INT. J. SYST.BACTERIOL. JANSSEN ET AL. carbon and energy sources and are variously fermented to L-lactate, formate, acetate, ethanol, and CO,. No respiratory metabolism occurs with oxygen, fumarate, nitrate, or sulfur compounds. Not generally polysaccharolytic, although some sugar polymers support growth. As determined by a 16s rDNA analysis, the genus Lactosphaera belongs to the group containing gram-positive lactic acid bacteria with low DNA G + C contents and is closely related to the genus Carnobacterium, but is distinguished by key signature nucleotides, by its coccus-shaped cells, and by its higher DNA G+C content (45 mol%, compared with 33 to 37 mol%). Description of Lactosphaera pusteurii comb. nov. Lactosphaera pasteurii (pas.teu'ri.i. M.L. gen. n. pasteurii, referring to Louis Pasteur, who probably first enriched and observed this bacterium during studies on tartrate fermentation). L. pasteurii cells are cocci that form pairs or small irregular packets of cells. The cells are 1.0 to 1.5 pm in diameter. No flagella are found. Growth occurs under an air atmosphere in complex media. Growth occurs at 0 to 42"C, at pH 5.5 to 9.0, and in the presence of NaCl concentrations up to 2% (wt/vol). Catalase and superoxide dismutase activities are absent. Biotin is required as a growth factor. Nitrate, sulfate, sulfite, thiosulfate, sulfur, and fumarate are not reduced. The presence of oxygen can result in a shift to more oxidized fermentation end products, but this is not coupled to energy conservation. No cytochromes are formed. The following growth substrates are utilized (L isomers of organic and amino acids and D isomers of sugars unless noted otherwise): L-tartrate, pyruvate, oxaloacetate, malate, citrate, mannitol, sorbitol, glucose, galactose, mannose, L-rhamnose, fructose, maltose, lactose, sucrose, cellobiose, raffinose, trehalose, sorbose, starch, oat spelt xylan, and laminarin (weak). D-Tartrate, rneso-tartrate, xylose, ribose, arabinose, malonate, succinate, ~~-3-hydroxybutyrate, lactate, amino acids, alcohols, chitin, gum karaya, carboxymethyl cellulose, amorphous cellulose, mannan, lichenan, gum locust bean, pullulan, arabinogalactan, and glycogen are not utilized. L-Tartrate, pyruvate, and citrate are fermented to acetate and formate (and CO,). Glucose and other carbohydrates are fermented to L-lactate, acetate, formate, and ethanol. Lactate production increases under acidic growth conditions and is mediated by a Fru-1,6-P2-activated LDH. LDH induction is controlled by pH. Succinate production and H, production are insignificant. Negative for oxidase activity, urease activity, indole production, sulfide production from cysteine, and gelatin hydrolysis. Esculin is hydrolyzed. No growth occurs on acetate agar (pH 5.4). The DNA base composition of strain K o T is~ 45~ mol% ~ G+C. The type strain is KoTa2 (= DSM 2381 = ATCC 35949, which was isolated from anoxic digestor sludge. ACKNOWLEDGMENTS We thank H. Hippe, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany, for providing unpublished data, the directors of the Thermophile Research Unit, University of Waikato, Hamilton, New Zealand, for access to facilities, and T. Ezaki, Gifu University School of Medicine, Gifu, Japan, for providing sequence data prior to publication. P.H.J. gratefully acknowledges a UGC scholarship (in New Zealand) and a fellowship from the Alexander-von-Humboldt-Stiftung (in Germany). 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